Inhibition of Arp2/3 Complex after ADP-Ribosylation of Arp2 by Binary Clostridioides Toxins.

Clostridioides bacteria are responsible for life threatening infections. Here, we show that in addition to actin, the binary toxins CDT, C2I, and Iota from Clostridioides difficile, botulinum, and perfrigens, respectively, ADP-ribosylate the actin-related protein Arp2 of Arp2/3 complex and its additional components ArpC1, ArpC2, and ArpC4/5. The Arp2/3 complex is composed of seven subunits and stimulates the formation of branched actin filament networks. This activity is inhibited after ADP-ribosylation of Arp2. Translocation of the ADP-ribosyltransferase component of CDT toxin into human colon carcinoma Caco2 cells led to ADP-ribosylation of cellular Arp2 and actin followed by a collapse of the lamellipodial extensions and F-actin network. Exposure of isolated mouse colon pieces to CDT toxin induced the dissolution of the enterocytes leading to luminal aggregation of cellular debris and the collapse of the mucosal organization. Thus, we identify the Arp2/3 complex as hitherto unknown target of clostridial ADP-ribosyltransferases.

Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G).

Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca2+-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca2+-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca2+-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca2+-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca2+-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.

De Novo Missense Mutations in TNNC1 and TNNI3 Causing Severe Infantile Cardiomyopathy Affect Myofilament Structure and Function and Are Modulated by Troponin Targeting Agents.

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in TNNC1 (p.cTnC-G34S) and TNNI3 (p.cTnI-D127Y) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit interactions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient's myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations.

Infantile restrictive cardiomyopathy: cTnI-R170G/W impair the interplay of sarcomeric proteins and the integrity of thin filaments.

TNNI3 encoding cTnI, the inhibitory subunit of the troponin complex, is the main target for mutations leading to restrictive cardiomyopathy (RCM). Here we investigate two cTnI-R170G/W amino acid replacements, identified in infantile RCM patients, which are located in the regulatory C-terminus of cTnI. The C-terminus is thought to modulate the function of the inhibitory region of cTnI. Both cTnI-R170G/W strongly enhanced the Ca2+-sensitivity of skinned fibres, as is typical for RCM-mutations. Both mutants strongly enhanced the affinity of troponin (cTn) to tropomyosin compared to wildtype cTn, whereas binding to actin was either strengthened (R170G) or weakened (R170W). Furthermore, the stability of reconstituted thin filaments was reduced as revealed by electron microscopy. Filaments containing R170G/W appeared wavy and showed breaks. Decoration of filaments with myosin subfragment S1 was normal in the presence of R170W, but was irregular with R170G. Surprisingly, both mutants did not affect the Ca2+-dependent activation of reconstituted cardiac thin filaments. In the presence of the N-terminal fragment of cardiac myosin binding protein C (cMyBPC-C0C2) cooperativity of thin filament activation was increased only when the filaments contained wildtype cTn. No effect was observed in the presence of cTn containing R170G/W. cMyBPC-C0C2 significantly reduced binding of wildtype troponin to actin/tropomyosin, but not of both mutant cTn. Moreover, we found a direct troponin/cMyBPC-C0C2 interaction using microscale thermophoresis and identified cTnI and cTnT, but not cTnC as binding partners for cMyBPC-C0C2. Only cTn containing cTnI-R170G showed a reduced affinity towards cMyBPC-C0C2. Our results suggest that the RCM cTnI variants R170G/W impair the communication between thin and thick filament proteins and destabilize thin filaments.

Interaction of isolated cross-linked short actin oligomers with the skeletal muscle myosin motor domain.

The cyclical interaction between F-actin and myosin in muscle cells generates contractile force. The myosin motor domain hydrolyses ATP, resulting in conformational changes that are amplified by the myosin lever arm that links the motor domain to the rod domain. Recent cryo-electron microscopic data have provided a clear picture of the myosin-ATP-F-actin complex, but structural insights into other stages of the myosin-actin interaction have been less forthcoming. To address this issue, we cross-linked F-actin subunits between Cys374 and Lys191, and separated them by gel filtration. Purified actin-dimers, -trimers and -tetramers retained the ability to polymerize and to stimulate myosin-subfragment 1 (myosin-S1) ATPase activity. To generate stable actin oligomer:myosin-S1 complexes, we blocked actin polymerization with gelsolin and Clostridium botulinum iota toxin-mediated ADP-ribosylation. After polymerization inhibition, actin-trimers and -tetramers retained the ability to stimulate the myosin-S1-ATPase, whereas the actin-dimer showed very little ATPase stimulation. We then analysed the stoichiometry and binding affinity of myosin-S1 to actin oligomers. Actin-trimers and -tetramers bound myosin-S1 in the absence of nucleotide; the trimer contains one myosin-S1 binding site. We calculated a dissociation constant (Kd ) of 1.1 × 10-10  m and 1.9 × 10-10  m for binding of native F-actin and the actin-trimer to myosin-S1, respectively. EM of the actin-trimer:myosin-S1 complex demonstrated the presence of single particles of uniform size. Image reconstruction allowed a reasonable fit of the actin-trimer and myosin-S1 into the obtained density clearly showing binding of one myosin-S1 molecule to the two long-pitch actins of the trimer, supporting the kinetic data.

Force-producing ADP state of myosin bound to actin.

Molecular motors produce force when they interact with their cellular tracks. For myosin motors, the primary force-generating state has MgADP tightly bound, whereas myosin is strongly bound to actin. We have generated an 8-Å cryoEM reconstruction of this state for myosin V and used molecular dynamics flexed fitting for model building. We compare this state to the subsequent state on actin (Rigor). The ADP-bound structure reveals that the actin-binding cleft is closed, even though MgADP is tightly bound. This state is accomplished by a previously unseen conformation of the ß-sheet underlying the nucleotide pocket. The transition from the force-generating ADP state to Rigor requires a 9.5° rotation of the myosin lever arm, coupled to a ß-sheet rearrangement. Thus, the structure reveals the detailed rearrangements underlying myosin force generation as well as the basis of strain-dependent ADP release that is essential for processive myosins, such as myosin V.

Structural basis for the allosteric interference of myosin function by reactive thiol region mutations G680A and G680V.

The cold-sensitive single-residue mutation of glycine 680 in the reactive thiol region of Dictyostelium discoideum myosin-2 or the corresponding conserved glycine in other myosin isoforms has been reported to interfere with motor function. Here we present the x-ray structures of myosin motor domain mutants G680A in the absence and presence of nucleotide as well as the apo structure of mutant G680V. Our results show that the Gly-680 mutations lead to uncoupling of the reactive thiol region from the surrounding structural elements. Structural and functional data indicate that the mutations induce the preferential population of a state that resembles the ADP-bound state. Moreover, the Gly-680 mutants display greatly reduced dynamic properties, which appear to be related to the recovery of myosin motor function at elevated temperatures.

Mechanism, regulation, and functional properties of Dictyostelium myosin-1B.

Myosin-1B is one of three long tailed class-1 myosins containing an ATP-insensitive actin-binding site in the tail region that are produced in Dictyostelium discoideum. Myosin-1B localizes to actin-rich structures at the leading edge of migrating cells where it has been implicated in the formation and retraction of membrane projections, the recycling of plasma membrane components, and intracellular particle transport. Here, we have used a combination of molecular engineering approaches to describe the kinetic and motile properties of the myosin-1B motor and its regulation by TEDS site phosphorylation. Our results show that myosin-1B is a low duty ratio motor and displays the fastest nucleotide binding kinetics of any of the Dictyostelium class-1 myosins studied so far. Different from Dictyostelium myosin-1D and myosin-1E, dephosphorylated myosin-1B is not inactivated but moves actin filaments efficiently, albeit at an up to 8-fold slower velocity in the in vitro motility assay. A further difference is that myosin-1B lacks the ability to switch between rapid movement and bearing tension upon physiological changes of free Mg2+ ions. In this respect, its motor properties appear to be more closely related to Dictyostelium myosin-2 and rabbit skeletal muscle myosin.

Functional characterization of the N-terminal region of myosin-2.

All class 2 myosins contain an N-terminal extension of approximately 80 residues that includes an Src homology 3 (SH3)-like subdomain. To explore the functional importance of this region, which is also present in most other myosin classes, we generated truncated constructs of Dictyostelium discoideum myosin-2. Truncation at position 80 resulted in the complete loss of myosin-2 function in vivo. Actin affinity was more than 80-fold, and the rate of ADP release approximately 40-fold decreased in this mutant. In contrast, a myosin construct that lacks only the SH3-like subdomain, corresponding to residues 33-79, displayed much smaller functional defects. In complementation experiments with myosin-2 null cells, this construct rescued myosin-2-dependent processes such as cytokinesis, fruiting body formation, and sporogenesis. An 8-fold reduction in motile activity and changes of similar extent in the affinity for ADP and filamentous actin indicate the importance of the SH3-like subdomain for correct communication between the functional regions within the myosin motor domain and suggest that local perturbations in this region can play a role in modulating myosin-2 motor activity.

The actin-binding cleft: functional characterisation of myosin II with a strut mutation.

The myosin cross-bridge has two essential properties: to undergo the "power stroke" and to bind and release from actin - both under control of ATP binding and hydrolysis. In the absence of ATP the cross-bridge binds to actin with high affinity: the binding of ATP causes rapid release of the cross-bridge from actin. The actin binding-site is split by a deep cleft that closes on strong binding to actin. The cleft is straddled by a short polypeptide known as the "strut". In the following we summarise the structural basis of the power stroke and the control of actin affinity and then present data on the effects on actin affinity of replacing the strut by a flexible linker.

Dictyostelium myosin-IE is a fast molecular motor involved in phagocytosis.

Class I myosins are single-headed motor proteins, implicated in various motile processes including organelle translocation, ion-channel gating, and cytoskeleton reorganization. Here we describe the cellular localization of myosin-IE and its role in the phagocytic uptake of solid particles and cells. A complete analysis of the kinetic and motor properties of Dictyostelium discoideum myosin-IE was achieved by the use of motor domain constructs with artificial lever arms. Class I myosins belonging to subclass IC like myosin-IE are thought to be tuned for tension maintenance or stress sensing. In contrast to this prediction, our results show myosin-IE to be a fast motor. Myosin-IE motor activity is regulated by myosin heavy chain phosphorylation, which increases the coupling efficiency between the actin and nucleotide binding sites tenfold and the motile activity more than fivefold. Changes in the level of free Mg(2+) ions, which are within the physiological range, are shown to modulate the motor activity of myosin-IE by inhibiting the release of adenosine diphosphate.

Changes in Mg2+ ion concentration and heavy chain phosphorylation regulate the motor activity of a class I myosin.

Class I myosins are single-headed motor proteins implicated in various motile processes including organelle translocation, ion channel gating, and cytoskeleton reorganization. Dictyostelium discoideum myosin-ID belongs to subclass 1alpha, whose members are thought to be tuned for rapid sliding. The direct analysis of myosin-ID motor activity is made possible by the production of single polypeptide constructs carrying an artificial lever arm. Using these constructs, we show that the motor activity of myosin-ID is activated 80-fold by phosphorylation at the TEDS site. TEDS site phosphorylation acts by stabilizing the actomyosin complex and increasing the coupling between actin binding and the release of hydrolysis products. A surprising effect of Mg(2+) ions on in vitro motility was discovered. Changes in the level of free Mg(2+) ions within the physiological range are shown to modulate motor activity by inhibiting ADP release. Our results indicate that higher concentrations of free Mg(2+) ions stabilize the tension-bearing actin myosin ADP state and shift the system from the production of rapid movement toward the generation of tension.

Molecular engineering of a backwards-moving myosin motor.

All members of the diverse myosin superfamily have a highly conserved globular motor domain that contains the actin- and nucleotide-binding sites and produces force and movement. The light-chain-binding domain connects the motor domain to a variety of functionally specialized tail domains and amplifies small structural changes in the motor domain through rotation of a lever arm. Myosins move on polarized actin filaments either forwards to the barbed (+) or backwards to the pointed (-) end. Here, we describe the engineering of an artificial backwards-moving myosin from three pre-existing molecular building blocks. These blocks are: a forward-moving class I myosin motor domain, a directional inverter formed by a four-helix bundle segment of human guanylate-binding protein-1 and an artificial lever arm formed by two alpha-actinin repeats. Our results prove that reverse-direction movement of myosins can be achieved simply by rotating the direction of the lever arm 180 degrees.

Mutations in the relay loop region result in dominant-negative inhibition of myosin II function in Dictyostelium.

Dominant-negative inhibition is a powerful genetic tool for the characterization of gene function in vivo, based on the specific impairment of a gene product by the coexpression of a mutant version of the same gene product. We describe the detailed characterization of two myosin constructs containing either point mutations F487A or F506G in the relay region. Dictyostelium cells transformed with F487A or F506G myosin are unable to undergo processes that require myosin II function, including fruiting-body formation, normal cytokinesis and growth in suspension. Our results show that the dominant-negative inhibition of myosin function is caused by disruption of the communication between active site and lever arm, which blocks motor activity completely, and perturbation of the communication between active site and actin-binding site, leading to an approximately 100-fold increase in the mutants' affinity for actin in the presence of ATP.

PEVK domain of titin: an entropic spring with actin-binding properties.

The PEVK domain of the giant muscle protein titin is a proline-rich sequence with unknown secondary/tertiary structure. Here we compared the force-extension behavior of cloned cardiac PEVK titin measured by single-molecule atomic force spectroscopy with the extensibility of the PEVK domain measured in intact cardiac muscle sarcomeres. The analysis revealed that cardiac PEVK titin acts as an entropic spring with the properties of a random coil exhibiting mechanical conformations of different flexibility. Since in situ, titin is in close proximity to the thin filaments, we also studied whether the PEVK domain of cardiac or skeletal titin may interact with actin filaments. Interaction was indeed found in the in vitro motility assay, in which recombinant PEVK titin constructs slowed down the sliding velocity of actin filaments over myosin. Skeletal PEVK titin affected the actin sliding to a lesser degree than cardiac PEVK titin. The cardiac PEVK effect was partially suppressed by physiological Ca(2+) concentrations, whereas the skeletal PEVK effect was independent of [Ca(2+)]. Cosedimentation assays confirmed the Ca(2+)-modulated actin-binding propensity of cardiac PEVK titin, but did not detect interaction between actin and skeletal PEVK titin. In myofibrils, the relatively weak actin-PEVK interaction gives rise to a viscous force component opposing filament sliding. Thus, the PEVK domain contributes not only to the extensibility of the sarcomere, but also affects contractile properties.

Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor.

Successful host cell invasion is a prerequisite for survival of the obligate intracellular apicomplexan parasites and establishment of infection. Toxoplasma gondii penetrates host cells by an active process involving its own actomyosin system and which is distinct from induced phagocytosis. Toxoplasma gondii myosin A (TgMyoA) is presumed to achieve power gliding motion and host cell penetration by the capping of apically released adhesins towards the rear of the parasite. We report here an extensive biochemical characterization of the functional TgMyoA motor complex. TgMyoA is anchored at the plasma membrane and binds a novel type of myosin light chain (TgMLC1). Despite some unusual features, the kinetic and mechanical properties of TgMyoA are unexpectedly similar to those of fast skeletal muscle myosins. Microneedle-laser trap and sliding velocity assays established that TgMyoA moves in unitary steps of 5.3 nm with a velocity of 5.2 microm/s towards the plus end of actin filaments. TgMyoA is the first fast, single-headed myosin and fulfils all the requirements for power parasite gliding.